3.1. ACP-93 induces apoptosis in both the securin-wild type and securin-null HCT116 cells
Fig. 1 shows the chemical structure of ACP-93. Previously, treatment with 2-32 μM ACP-93 for 24 h induced cytotoxicity in both the securin-wild type and securin-null
HCT116 cells [87]. We have further assessed the apoptosis induction by ACP-93 in the securin-wild type and securin-null HCT116 cells in this study. The ACP-93 treated cells were analyzed by Annexin V-FITC-PI staining. The Annexin V+/PI- cells (early apoptosis) and Annexin V+/PI+ cells (late apoptosis) were increased following treatment with ACP-93 at 8 μM to 32 μM 24 h in both the securin-wild type and securin-null HCT116 cells (Fig. 2A). ACP-93 (32 μM for 24 h) significantly induced apoptosis level at average of 13.48% in the securin-wild type HCT116 cells and 11.82% in the securin-null HCT116 cells (Fig. 2B).
3.2. ACP-93 increases the protein levels of active caspase 3 and caspase 8 and the protein cleavage of PARP in the securin-wild type and securin-null HCT116 cells
To examine the apoptotic-regulated protein whether involve in ACP-93-induced apoptosis, caspases 3, caspase 8, PARP and bax proteins were analyzed by Western blot.
The active form of caspase 3 (17 kDa and 12 kDa) protein was induced following treatment with 4-32 μM ACP-93 for 24 h in both the securin-wild type and securin-null
HCT116 cells (Fig. 3A). Also, the cleaved form of PARP (89 kDa) protein was elicited after treatment with 4-32 μM ACP-93 for 24 h. ACP-93 (32 μM for 24 h) significantly induced the active form of caspase 3 and the cleaved-PARP in the securin-wild type and securin-null HCT116 cells (Fig. 3B). However, the bax protein expression was not altered following ACP-93 treatment in the securin-wild type and securin-null HCT116 cells (Fig. 3A). To analyze the extrinsic apoptosis pathway whether regulated ACP-93-induced apoptosis, the active form of caspase 8 protein was analyzed by Western blot. As shown in fig. 4A, treatment with 4-32 μM ACP-93 for 24 h increased active caspase 8 protein levels. ACP-93 (32 μM for 24 h) significantly increased the active form of caspase 8 in both the securin-wild type and securin-null HCT116 cells (Fig. 4B). Actin was used as a loading control protein that was not altered by ACP-93.
3.3. ACP-93 induces γ-H2AX and ATF-3 proteins expression in the securin-wild type and securin-null HCT116 cells
To analyze the effect of ACP-93 on the apoptotic-regulated proteins expression, the phosphorylated H2AX and ATF-3 were analyzed to Western blot. As shown in fig.
5A, the protein expression of γ-H2AX was induced by ACP-93. Treatment with 32 μM ACP-93 for 24 h significantly induced protein expression in both the securin-wild type and securin-null HCT116 cells (Fig. 5B). Furthermore, the ATF-3 protein was also
induced following 4-32 μM ACP-93 for 24 h (Fig. 6A). ACP-93 significantly increased
F- protein expression in both the securin-wild type and securin-null HCT116 cells (Fig. 6B). Actin was used as a loading control protein that was not altered by ACP-93.
3.4. ACP-93 induces ROS generation in the securin-wild type and securin-null HCT116 cells
The intercellular ROS level of the securin-wild type and securin-null HCT116 cells were determined by flow cytometry and staining with fluorescent dye H2DCF-DA.
The DCF fluorescence intensity was increased after 4-32 μM ACP-93 treatment in both the securin-wild type and securin-null HCT116 cells (Fig. 7).
3.5. ACP-93 inhibits cell proliferation and increases sub-G1 fractions in the securin-wild type and securin-null HCT116 cells
The cell proliferation was inhibited by ACP-93 (32 μM) when the ACP-93-treated cells were re-cultured for 2-6 days (Fig. 8). To determine the possible involvement of ACP-93 in the regulation of cell cycle progression, the effect of ACP-93 on the securin-wild type and securin-null HCT116 cells were analyzed by flow cytometry (Fig. 9A). ACP-93 did not significantly alter the fractions of G1, S andG2/M phases in the securin-wild type and securin-null HCT116 cells. However, it markedly increased the fractions of sub-G phase in both the securin-wild type and securin-null
HCT116 cells (Fig. 9B). Treatment with 32 μM ACP-93 for 24 h increased sub-G1
fractions by about 7.7% and 9% in the securin-wild type and securin-null HCT116 cells, respectively.
3.6. ACP-93 induces anti-apoptotic protein expression in colon cancer cells
To investigate whether the anti-apoptotic proteins in regulating apoptosis by ACP-93, AKT, bcl-2 and survivin protein levels were analyzed by Western blot.
Treatment with 4-32 μM ACP-93 for 24 h increased phospho-AKT (ser473), bcl-2 and survivin protein expression (Fig. 10). Total AKT protein expression did not alter by ACP-93. The protein levels of phospho-AKT (ser473), bcl-2 and survivin were increased at 4-24 h whereas decreased at 36 h by 32 μM ACP-93 treatment (Fig. 11).
Actin was used as a loading control protein that did not alter by ACP-93. Besides, co-treatment with wortmannin significantly increased the ACP-93-induced cytotoxicity in the securin-wild type HCT116 cells (Fig. 12).
3.7. Securin promotes tumorigenesis of xenograft human colon tumors in nude mice
To study the role of securin in tumorigenesis, the securin-wild type and securin-null colon tumor xenograft nude mice were analyzed. The tumor developed in
four-week-old nude mice and each mouse received 2 × 106 cells by subcutaneously injection. The tumors of xenograft mice were growth to average 1995.5 mm3 in the securin-wild type groups and 910.4 mm3 in the securin-null groups after inoculation for 60 days (Fig. 13A). The solid securin-wild type and securin-null tumors were separated from mice (Fig. 13B).
3.8. ACP-93 inhibits tumorigenesis of xenograft human colon tumors in SCID and nude mice
To evaluate the anti-tumorigenesis effect of ACP-93, the colon tumor xenograft in SCID mice and nude mice were analyzed. In the SCID mice model, the tumors of xenograft mice were growth to 595 mm3 in control groups and no visible tumors in ACP-93 pre-treatment xenograft mice until 40 days (Fig. 14A). Post-treatment of ACP-93 (30 mg/kg) in colon tumor xenograft SCID mice reveal inhibitory effect on tumorigenesis comparing to the control groups (Fig. 14B). The tumors of xenograft mice were growth to average 419 mm3 in control groups and 296 mm3 in ACP-93 treated groups after inoculation for 28 days. In addition, the nude mice model was investigated. Post-treatment of ACP-93 (30 mg/kg) in colon tumor xenograft nude mice reveal inhibitory effect on tumorigenesis comparing to the control groups (Fig. 15). The tumors of xenograft mice were growth to average 140 mm3 in control groups and 47.8
mm3 in ACP-93 treated groups after inoculation for 32 days.